John Crum

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilins SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Depolarization Redistributes Synaptic Membrane and Creates a Gradient of Vesicles on the Synaptic Body at a Ribbon Synapse

We used electron tomography of frog saccular hair cells to reconstruct presynaptic ultrastructure at synapses specialized for sustained transmitter release. Synaptic vesicles at inhibited synapses were abundant in the cytoplasm and covered the synaptic body at high density. Continuous maximal stimulation depleted 73% of the vesicles within 800 nm of the synapse, with a concomitant increase in surface area of intracellular cisterns and plasmalemmal infoldings. Docked vesicles were depleted 60%-80% regardless of their distance from the active zone. Vesicles on the synaptic body were depleted primarily in the hemisphere facing the plasmalemma, creating a gradient of vesicles on its surface. We conclude that formation of new synaptic vesicles from cisterns is rate limiting in the vesicle cycle.

NF-M is an essential target for the myelin-directed “outside-in” signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an “outside-in” signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

Direct Restriction of Virus Release and Incorporation of the Interferon-Induced Protein BST-2 into HIV-1 Particles

Investigation of the Vpu protein of HIV-1 recently uncovered a novel aspect of the mammalian innate response to enveloped viruses: retention of progeny virions on the surface of infected cells by the interferon-induced, transmembrane and GPI-anchored protein BST-2 (CD317; tetherin). BST-2 inhibits diverse families of enveloped viruses, but how it restricts viral release is unclear. Here, immuno-electron microscopic data indicate that BST-2 is positioned to directly retain nascent HIV virions on the plasma membrane of infected cells and is incorporated into virions. Virion-incorporation was confirmed by capture of infectivity using antibody to the ectodomain of BST-2. Consistent with a direct tethering mechanism, we confirmed that proteolysis releases restricted virions and further show that this removed the ectodomain of BST-2 from the cell surface. Unexpectedly, enzymatic cleavage of GPI anchors did not release restricted virions, weighing against models in which individual BST-2 molecules span the virion and host cell membranes. Although the exact molecular topology of restriction remains unsolved, we suggest that the incorporation of BST-2 into viral envelopes underlies its broad restrictive activity, whereas its relative exclusion from virions and sites of viral assembly by proteins such as HIV-1 Vpu may provide viral antagonism of restriction.

Electron tomographic analysis of synaptic ultrastructure.

Synaptic function depends on interactions among sets of proteins that assemble into complex supramolecular machines. Molecular biology, electrophysiology, and live‐cell imaging studies have provided tantalizing glimpses into the inner workings of the synapse, but fundamental questions remain regarding the functional organization of these “nano‐machines.” Electron tomography reveals the internal structure of synapses in three dimensions with exceptional spatial resolution. Here we report results from an electron tomographic study of axospinous synapses in neocortex and hippocampus of the adult rat, based on aldehyde‐fixed material stabilized with tannic acid in lieu of postfixation with osmium tetroxide. Our results provide a new window into the structural basis of excitatory synaptic processing in the mammalian brain. J. Comp. Neurol. 520:2697–2711, 2012.

Ultrastructural organization of lamprey reticulospinal synapses in three dimensions

The giant reticulospinal synapse in lamprey provides a unique model to study synaptic vesicle traffic. The axon permits microinjections, and the active zones are often separated from each other, which makes it possible to track vesicle cycling at individual release sites. However, the proportion of reticulospinal synapses with individual active zones (“simple synapses”) is unknown and a quantitative description of their organization is lacking. Here, we report such data obtained by serial section analysis, intermediate‐voltage electron microscopy, and electron tomography. The simple synapse was the most common type (78%). It consisted of one active zone contacting one dendritic process. The remaining synapses were “complex,” mostly containing one vesicle cluster and two to three active zones synapsing with distinct dendritic shafts. Occasional axosomatic synapses with multiple active zones forming synapses with the same cell were also observed. The vast majority of active zones in all synapse types contained both chemical and electrotonic synaptic specializations. Quantitative analysis of simple synapses showed that the majority had active zones with a diameter of 0.8–1.8 μm. The number of synaptic vesicles and the height of the vesicle cluster in middle sections of serially cut synapses correlated with the active zone length within, but not above, this size range. Electron tomography of simple synapses revealed small filaments between the clustered synaptic vesicles. A single vesicle could be in contact with up to 12 filaments. Another type of filament, also associated with synaptic vesicles, emerged from dense projections. Up to six filaments could be traced from one dense projection. J. Comp. Neurol. 450:167–182, 2002.

Electron tomography, three-dimensional Fourier analysis and colour prediction of a three-dimensional amorphous biophotonic nanostructure

Organismal colour can be created by selective absorption of light by pigments or light scattering by photonic nanostructures. Photonic nanostructures may vary in refractive index over one, two or three dimensions and may be periodic over large spatial scales or amorphous with short-range order. Theoretical optical analysis of three-dimensional amorphous nanostructures has been challenging because these structures are difficult to describe accurately from conventional two-dimensional electron microscopy alone. Intermediate voltage electron microscopy (IVEM) with tomographic reconstruction adds three-dimensional data by using a high-power electron beam to penetrate and image sections of material sufficiently thick to contain a significant portion of the structure. Here, we use IVEM tomography to characterize a non-iridescent, three-dimensional biophotonic nanostructure: the spongy medullary layer from eastern bluebird Sialia sialis feather barbs. Tomography and three-dimensional Fourier analysis reveal that it is an amorphous, interconnected bicontinuous matrix that is appropriately ordered at local spatial scales in all three dimensions to coherently scatter light. The predicted reflectance spectra from the three-dimensional Fourier analysis are more precise than those predicted by previous two-dimensional Fourier analysis of transmission electron microscopy sections. These results highlight the usefulness, and obstacles, of tomography in the description and analysis of three-dimensional photonic structures.

Phosphorylation of Highly Conserved Neurofilament Medium KSP Repeats Is Not Required for Myelin-Dependent Radial Axonal Growth

Neurofilament medium (NF-M) is essential for the acquisition of normal axonal caliber in response to a myelin-dependent “outside-in” trigger for radial axonal growth. Removal of the tail domain and lysine-serine-proline (KSP) repeats of NF-M, but not neurofilament heavy, produced axons with impaired radial growth and reduced conduction velocities. These earlier findings supported myelin-dependent phosphorylation of NF-M KSP repeats as an essential component of axonal growth. As a direct test of whether phosphorylation of NF-M KSP repeats is the target for the myelin-derived signal, gene replacement has now been used to produce mice in which all serines of NF-Ms KSP repeats have been replaced with phosphorylation-incompetent alanines. This substitution did not alter accumulation of the neurofilaments or their subunits. Axonal caliber and motor neuron conduction velocity of mice expressing KSP phospho-incompetent NF-M were also indistinguishable from wild-type mice. Thus, phosphorylation of NF-M KSP repeats is not an essential component for the acquisition of normal axonal caliber mediated by myelin-dependent outside-in signaling.

Three-dimensional reconstruction of the amphid sensilla in the microbial feeding nematode, Acrobeles complexus (nematoda: Rhabditida)

Amphid sensilla are the primary olfactory, chemoreceptive, and thermoreceptive organs in nematodes. Their function is well described for the model organism Caenorhabditis elegans, but it is not clear to what extent we can generalize these findings to distantly related nematodes of medical, economic, and agricultural importance. Current detailed descriptions of anatomy and sensory function are limited to nematodes that recent molecular phylogenies would place in the same taxonomic family, the Rhabditidae. Using serial thin‐section transmission electron microscopy, we reconstructed the anatomy of the amphid sensilla in the more distantly related nematode, Acrobeles complexus (Cephalobidae). Amphid structure is broadly conserved in number and arrangement of cells. Details of cell anatomy differ, particularly for the sensory neurite termini. We identify an additional sensory neuron not found in the amphid of C. elegans and propose homology with the C. elegans interneuron AUA. Hypotheses of homology for the remaining sensory neurons are also proposed based on comparisons between C. elegans, Strongyloides stercoralis, and Haemonchus contortus. J. Comp. Neurol. 512:271–281, 2009.

The cytoplasmic accumulations of the cataract-associated mutant, Connexin50P88S, are long-lived and form in the endoplasmic reticulum

Mutant connexins have been linked to hereditary congenital cataracts. One such mutant causes a proline-to-serine substitution at position 88 in human connexin 50 (CX50P88S). In transfected cells, CX50P88S does not form gap junctions, but localizes in cytoplasmic multilamellar structures. We studied the dynamics of formation and the stability of these structures in HeLa cells stably transfected with CX50P88S containing a tetracysteine motif appended to its C-terminus (HeLa-CX50P88S(Cys)(4) cells). The tetracysteine motif binds the membrane-permeable biarsenical compounds, FlAsH and ReAsH, which become fluorescent upon binding allowing detection of CX50P88S(Cys)(4) by fluorescence microscopy or by transmission electron microscopy after the ReAsH-driven fluorescent photoconversion of diaminobenzidine. CX50P88S structures were long-lived. Pulse labeling of HeLa-CX50P88S(Cys)(4) cells with FlAsH followed by a chase and ReAsH labeling showed a differential distribution of the labels, with older CX50P88S surrounded by newly synthesized protein. Formation of CX50P88S accumulations was not affected by treatments that block ER-to-Golgi transport. Transmission electron microscopy and tomographic reconstruction revealed that CX50P88S accumulations corresponded to closely apposed circular or semicircular membrane stacks that were sometimes continuous with the rough endoplasmic reticulum. These results suggest that CX50P88S accumulations originate from the rough endoplasmic reticulum and that mutant protein is sequentially added resulting in long-lived cytoplasmic particles. The persistence of these particles in the lens may cause light scattering and the pulverulent cataracts observed in affected individuals.